Human endometrial specific steroid-binding factor I, II and III

ABSTRACT

The invention relates to hESF I, II and III polypeptides, polynucleotides encoding the polypeptides, methods for producing the polypeptides, in particular by expressing the polynucleotides, and agonists and antagonists of the polypeptides. The invention further relates to methods for utilizing such polynucleotides, polypeptides, agonists and antagonists for applications, which relate, in part, to research, diagnostic and clinical arts.

This application is a divisional and claims priority under 35 U.S.C.§120 to application Ser. No. 09/583,169, filed May 30, 2000 (now issuedU.S. Pat. No. 6,338,948 B1), which is a divisional of application Ser.No. 09/263,810, filed Mar. 8, 1999 (now issued U.S. Pat. No. 6,174,992B1), which is a divisional of application Ser. No. 08/821,451, filedMar. 21, 1997 (now issued U.S. Pat. No. 6,066,724), which claimspriority under 35 U.S.C. §119(e) to Provisional Application Serial No.60/014,724, filed Mar. 21, 1996, all herein incorporated by reference intheir entirety.

This invention relates, in part, to newly identified polynucleotides andpolypeptides; variants and derivatives of the polynucleotides andpolypeptides; processes for making the polynucleotides and thepolypeptides, and their variants and derivatives; agonists andantagonists of the polypeptides; and uses of the polynucleotides,polypeptides, variants, derivatives, agonists and antagonists. Inparticular, in these and in other regards, the invention relates topolynucleotides and polypeptides of human endometrial specificsteroid-binding factor I, II and III, sometimes hereinafter referred toas “hESF I, II and III”.

BACKGROUND OF THE INVENTION

The regulation of cells and tissues is controlled by autocrine andparacrine factors, such as systemic hormones and factors that modulateor mediate the action of hormones.

Many peptides, expressed locally, can influence certain biologicalactivity in the mammalian system and are very important in theregulation of cells of the epithelium. These factors largely have notbeen identified or characterized particularly not in humans.

A few factors that play a role in the regulation of functions of thelung and uterus, both adult and fetal, have been identified in non-humanorganisms. One such factor is mammalian CC10, i.e., human, rat andrabbit CClO. (Wolf, M. et al., Human Molecular Genetics, 1(6):371-378(1992)). Clara Cell 10 kDa secretory protein (CC10) which is also calledClara Cell 17 kDa protein, is a homodimer consisting of 8.5 kDa monomersthat are joined by two disulfide bonds (Umland, T. C. et al., J. Mol.Biol., 224:441-448 (1992)). It is the predominant secreted protein oflung Clara cells which are the lining of the bronchiolar epithelium(Singh, G. and Katyal, S. L., J. Histochem. Cytochem., 32:49-54 (1984)).The physiological role of the protein is not yet completely understood.It has been reported that CC10 specifically bindsmethylsulfonyl-polychlorated biphenyls (PCBs) (Nordlund, Moler, L. etal., J. Biol. Chem., 265:12690-12693 (1990)) and inhibits phospholipaseA₂ (Singh, G. et al., Biochem. Biophys. Acta, 1039:348-355 (1990)). Inthe last few years the sequences of rat (Katyal, S. L. et al., Proc.Respir. Res., 25:29-35 (1990); and Hagen, G. et al., Nucleic Acids Res.,18:2939-2946 (1990)), and human (Singh, G. et al., Biochem. Biophys.Acta, 950:329-337 (1988) CC10 cDNAs have been reported. cDNAs, and thederived amino acid sequences, show striking homologies to ratuteroglobin (Singh, G. et al., Biochem. Biophys. Acta, 1039:348-355(1990); and Hagen, G. et al., Nucleic Acids Res., 18:2939-2946 (1990)).

Like CC10, rat uteroglobin is a covalently bound homodimer whose threedimensional structure is well known (Morize, I. et al., J. Mol. Biol.,194:725-739 (1987). Uteroglobin expression in rabbits has beenoriginally reported in the uterus during the preimplantation phase(Beier, H. M., Biochem. Biophys. Acta, 160:289-290 (1968)). Morerecently, the protein was also detected in oviduct (Kirchner, C., CellTissue Res., 170:490-492 (1976)), male genital organs (Beier, H. M. etal., Cell Tissue Res., 165:1-11 (1975)), esophagus (Noske, I. G. andFeigelson, M., Biol. Reprod., 15:704-713 (1976)) and lung (Noske, supra;and Torkkeli, T. et al., Biochem. Biophys. Acta, 544:578-592 (1978)).

In vitro, several distinct properties of uteroglobin have beendescribed. Soon after its discovery it could be shown that the steroidhormone progesterone is specifically bound by the protein (Beato, M. andBaier, R., Biochem. Biophys. Acta, 392:346-356 (1975); and Beato, M. etal., J. Steriod Biochem., 8:725-730 (1977)) Therefore, rabbituteroglobin was believed to be a potential carrier or scavenger ofprogesterone that regulates the progesterone concentration in theendometrium (Atger, M. et al., J. Steroid Biochem., 13:1157-1162(1980)). It has also been shown to specifically bind certainmethylsulfonyl metabolites of polychlorinated biphenyls with even higheraffinity than progesterone (Gillner, M. et al., J. Steroid Biochem.,31:27-33 (1988)). Furthermore, uteroglobin has been found to inhibitphospholipase A₂. The relationships of all these properties and theirphysiological significance is still not understood and remains largely amatter of speculation.

The rat CC10 mRNA is expressed like rat uteroglobin not only in lung butalso in the esophagus as well in uteri of estrogen and progesteronetreated female rats (Hagen, G. 1990 supra) suggesting that rat CC10 isthe rat counterpart of rat uteroglobin (see in general Wolf, M. et al.,Human Molecular Genetics, 1(6):371-378 (1992)).

Human CC10 expression is abundant in non-neoplastic human lung, and itis detectable in tumors in corresponding cell lines at markedly lowerlevels (Broers, J. L. V. et al., Lab. Invest., 66:337-346 (1992);Linnoila, R. I. et al., Amer. J. Clin. Pathol., 90:1-12 (1988)). CC10levels were also significantly lower in serum and bronchoalveolar lavagespecimens obtained from smokers and lung cancer patients compared withspecimens from healthy non-smokers (Bernard, A. et al., Europ. Resp. J.,5:1231-1238 (1992)).

These findings suggest the expression of CC10 mRNA becomes altered indistinct lung compartments and may implicate a role for CC10 in thedevelopment of pulmonary carcinomas (Jensen, S. M. et al., Int. J.Cancer, 58:629-637 (1994).

Some of the biological properties of UG, such as masking theantigenicity of blastomers (Mukherjee, A. B., et al., Med. Hypotheses,6:1043-1055 (1980)) and epididymal spermatozoa (Mukherjee, D. C., etal., Science (Wash. D.C.), 219:989-991 (1983)), inhibition of monocyteand neutrophil chemotaxis and phagocytosis in vitro (Schiffman, E. V.,et al., Agents Actions Suppl., 12:106-120 (1983)), and inhibition ofADP- and thrombin-induced (but not of arachidonic acid-induced) plateletaggregation (Manjunath, R., et al., Biochem. Pharmacol., 36:741-746(1987)), may be due, at least in part, to the potent inhibitory effectof this protein on PLA₂ activity (Levin, S. W., et al., Life Sci.,38:1813-1819 (1986)). A nonapeptide derived from the amino acid sequenceof α-helix-3 of UG monomer (residues 39-47) possesses all the biologicalproperties of the intact protein and has been identified as an activesite of UG responsible for its PLA₂-inhibitory and antiinflammatoryactivities (Miele, L., et al., Nature (Lond.), 335:726-730 (1988)).

It has been indicated that cc10kD-specific transcripts are present inseveral nonrespiratory human organs and tissues. By using an antibody torabbit UG, a UG-like immunoreactivity in human endometrium (Kikukawa,T., et al., J. Clin. Endocrinol. Metab., 67:315-321 (1988)), prostate(Manyak, M. J., et al., J. Urol., 140:176-182 (1988)), and respiratorytract (Dhanireddy, R., et al., Biochem. Biophys. Res. Commun.,152:1447-1454 (1988)), has been described.

Recently, the cDNA (Singh, G., et al., Biochem. BioThys. Acta,950:329-337 (1988)) and the 5′ regions (Wolf, M., et al., Human Mol.Genet., 1:371-378 (1992)) of the gene encoding human uteroglobin (hUG),a counterpart of rabbit UG (rUG), has been characterized. Human UG orClara cell 10-kD protein has 61.5% amino acid sequence identity with rUG(Singh, G., et al., Biochem. Biophys. Acta, 950:329-337 (1988)), 54.2%similarity with rat UG (Singh, G., et al., Biochem. Biophys. Acta,1039:348-355 (1990)), and 52.8% with mouse UG (Singh, G., et al., Exp.Lung Res., 19:67-75 (1993)). Although this protein was originallydiscovered in the alveolar Clara cells (Singh, G., et al., J.Histochem., 36:73-80 (1988)) it is detectable in many extrapulmonarytissues similar to the ones in which rUG is expressed (Peri, A., et al.,DNA Cell Biol., 5:495-503 (1994)) and this expression is induced byprogesterone. It appear that some of the biological properties of hUGare virtually identical to rUG (Mantile, G., et al., J. Biol. Chem.,27:20343-20351 (1993)).

It has been reported that UG in the rabbit uterine fluid is firstdetectable on day 3 of pregnancy, and peak level is reached on day 5(for a review see Miele, L., et al., Endocr. Rev., 8:474-490 (1987)).UG, by inhibiting PLA₂ activity, may down-regulate the production ofproinflammatory lipid mediators, which promote contraction and motilityof the uterine smooth muscle. Therefore, it is suggested that UGfacilitates the maintenance of myometrial quiescence during gestation.

There is a clear need in the art to further isolate and characterizeproteins which are homologues of mammalian Clara cell 10 kDa secretoryprotein and rat prostatic steroid-binding protein. The genes and geneproducts of the present invention display homology to the rat prostaticsteroid-binding protein and Clara cell 10 kDa secretory protein.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide polypeptides, inter alia, that have been identified as novelhESF I, II and III by homology between the amino acid sequence set outin FIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and 6) and known amino acidsequences of other proteins such as rat prostatic steroid-bindingprotein.

It is a further object of the invention, moreover, to providepolynucleotides that encode hESF I, II and III, particularlypolynucleotides that encode the polypeptides herein designated hESF I,II and III.

In a particularly preferred embodiment of this aspect of the inventionthe polynucleotides comprise the regions encoding human hESF I, II andIII in the sequence set out in FIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and 6).

In accordance with this aspect of the present invention there isprovided isolated nucleic acid molecules encoding mature polypeptidesexpressed by the human cDNA contained in ATCC Deposit No. 97401 (ESF I),97402 (ESF II) and 97403 (ESF III).

In accordance with this aspect of the invention there are providedisolated nucleic acid molecules encoding human hESF I, II and III,including mRNAs, cDNAs, genomic DNAs and, in further embodiments of thisaspect of the invention, biologically, diagnostically, clinically ortherapeutically useful variants, analogs or derivatives thereof, orfragments thereof, including fragments of the variants, analogs andderivatives.

Among the particularly preferred embodiments of this aspect of theinvention are naturally occurring allelic variants of human hESF I, IIand III.

It also is an object of the invention to provide hESF I, II and IIIpolypeptides, particularly human hESF I, II and III polypeptides, thattreat and/or prevent inflammation, asthma, rhinitis, cystic fibrosis,airway disease, neoplasia, atopy, inhibit phospholipase A₂ activity,bind polychlorinated biphenyls, reduce foreign protein antigenicity,inhibit monocyte and neutrophil chemotaxis and phagocytosis, inhibitplatelet aggregation, regulate eicosanoid levels in the human uterus andcontrol the growth of endometrial cells.

In accordance with this aspect of the invention there are provided novelpolypeptides of human origin referred to herein as hESF I, II and III aswell as biologically, diagnostically or therapeutically usefulfragments, variants and derivatives thereof, variants and derivatives ofthe fragments, and analogs of the foregoing.

Among the particularly preferred embodiments of this aspect of theinvention are variants of human hESF I, II and III encoded by naturallyoccurring alleles of the human hESF I, II and III genes.

It is another object of the invention to provide a process for producingthe aforementioned polypeptides, polypeptide fragments, variants andderivatives, fragments of the variants and derivatives, and analogs ofthe foregoing. In a preferred embodiment of this aspect of the inventionthere are provided methods for producing the aforementioned hESF I, IIand III polypeptides comprising culturing host cells having expressiblyincorporated therein an exogenously-derived human hESF I, II or IIIencoding polynucleotide under conditions for expression of human hESF I,II and III in the host and then recovering the expressed polypeptides.

In accordance with another object of the invention there are providedproducts, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for research,biological, clinical and therapeutic purposes, inter alia.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods, interalia, for, among other things: assessing hESF I, II and III expressionin cells by determining hESF I, II and III polypeptides or hESF I, IIand III-encoding mRNA; expressing hESF I, II and III in vitro, ex vivoor in vivo by exposing cells to hESF I, II and III polypeptides orpolynucleotides as disclosed herein; assaying genetic variation andaberrations, such as defects, in hESF I, II and III genes; andadministering a hESF I, II and III polypeptide or polynucleotide to anorganism to augment hESF I, II and III function or remediate hESF I, IIand III dysfunction.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided probes that hybridize tohuman hESF I, II and III sequences.

In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against hESF I, II and IIIpolypeptides. In certain particularly preferred embodiments in thisregard, the antibodies are highly selective for human hESF I, II andIII.

In accordance with another aspect of the present invention, there areprovided hESF I, II and III agonists. Among preferred agonists aremolecules that mimic hESF I, II and III, that bind to hESF I, II andIII-binding molecules or receptor molecules, and that elicit or augmenthESF I, II and III-induced responses. Also among preferred agonists aremolecules that interact with hESF I, II and III polypeptides, or withother modulators of hESF I, II and III activities, and therebypotentiate or augment an effect(s) of hESF I, II and III.

In accordance with yet another aspect of the present invention, thereare provided hESF I, II and III antagonists. Among preferred antagonistsare those which mimic hESF I, II and III so as to bind to hESF I, II andIII receptors or binding molecules but not elicit a hESF I, II andIII-induced response or more than one hESF I, II and III-inducedresponse or which prevent expression of hESF I, II and III. Also amongpreferred antagonists are molecules that bind to or interact with hESFI, II and III so as to inhibit an effect(s) of hESF I, II and III.

The agonists and antagonists may be used to mimic, augment or inhibitthe action of hESF I, II and III polypeptides. They may be used, forinstance, to treat and/or prevent an inherited susceptibility to asthma.

In a further aspect of the invention there are provided compositionscomprising a hESF I, II or III polynucleotide or a hESF I, II or IIIpolypeptide for administration to cells in vitro, to cells ex vivo andto cells in vivo, or to a multicellular organism. In certainparticularly preferred embodiments of this aspect of the invention, thecompositions comprise a hESF I, II or III polynucleotide for expressionof a hESF I, II or III polypeptide in a host organism for treatment ofdisease. Particularly preferred in this regard is expression in a humanpatient for treatment of a dysfunction associated with aberrantendogenous activity.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from, the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIG. 1 shows the nucleotide and deduced amino acid sequence of humanhESF I.

FIG. 2 shows the nucleotide and deduced amino acid sequence of humanhESF II.

FIG. 3 shows the nucleotide and deduced amino acid sequence of humanhESF III.

FIG. 4 shows the regions of similarity between amino acid sequences ofhESF I and rat prostatic steroid-binding protein polypeptides (SEQ IDNO:25).

FIG. 5 shows the regions of similarity between amino acid sequences ofhESF II and rat prostatic steroid-binding protein polypeptides (SEQ IDNO:26).

FIG. 6 shows the regions of similarity between amino acid sequences ofhESF III and rat prostatic steroid-binding protein polypeptides (SEQ IDNO:27).

FIG. 7 shows structural and functional features of hESF I deduced by theindicated techniques, as a function of amino acid sequence.

FIG. 8 shows structural and functional features of hESF II deduced bythe indicated techniques, as a function of amino acid sequence.

FIG. 9 shows structural and functional features of hESF III deduced bythe indicated techniques, as a function of amino acid sequence.

GLOSSARY

The following illustrative explanations are provided to facilitateunderstanding of certain terms used frequently herein, particularly inthe examples. The explanations are provided as a convenience and are notlimitative of the invention.

DIGESTION of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes referred to herein are commerciallyavailable and their reaction conditions, cofactors and otherrequirements for use are known and routine to the skilled artisan.

For analytical purposes, typically, 1 μg of plasmid or DNA fragment isdigested with about 2 units of enzyme in about 20 μl of reaction buffer.For the purpose of isolating DNA fragments for plasmid construction,typically 5 to 50 μg of DNA are digested with 20 to 250, units of enzymein proportionately larger volumes.

Appropriate buffers and substrate amounts for particular restrictionenzymes are described in standard laboratory manuals, such as thosereferenced below, and they are specified by commercial suppliers.

Incubation times of about 1 hour at 37° C. are ordinarily used, butconditions may vary in accordance with standard procedures, thesupplier's instructions and the particulars of the reaction. Afterdigestion, reactions may be analyzed, and fragments may be purified byelectrophoresis through an agarose or polyacrylamide gel, using wellknown methods that are routine for those skilled in the art.

GENETIC ELEMENT generally means a polynucleotide comprising a regionthat encodes a polypeptide or a region that regulates transcription ortranslation or other processes important to expression of thepolypeptide in a host cell, or a polynucleotide comprising both a regionthat encodes a polypeptide and a region operably linked thereto thatregulates expression.

Genetic elements may be comprised within a, vector that replicates as anepisomal element; that is, as a molecule physically independent of thehost cell genome. They may be comprised within mini-chromosomes, such asthose that arise during amplification of transfected DNA by methotrexateselection in eukaryotic cells. Genetic elements also may be comprisedwithin a host cell genome; not in their natural state but, rather,following manipulation such as isolation, cloning and introduction intoa host cell in the form of purified DNA or in a vector, among others.

ISOLATED means altered “by the hand of man” from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both.

For example, a naturally occurring polynucleotide or a polypeptidenaturally present in a living animal in its natural state is not“isolated,” but the same polynucleotide or polypeptide separated fromthe coexisting materials of its natural state is “isolated”, as the termis employed herein. For example, with respect to polynucleotides, theterm isolated means that it is separated from the chromosome and cell inwhich it naturally occurs.

As part of or following isolation, such polynucleotides can be joined toother polynucleotides, such as DNAs, for mutagenesis, to form fusionproteins, and for propagation or expression in a host, for instance. Theisolated polynucleotides, alone or joined to other polynucleotides suchas vectors, can be introduced into host cells, in culture or in wholeorganisms. Introduced into host cells in culture or in whole organisms,such DNAs still would be isolated, as the term is used herein, becausethey would not be in their naturally occurring form or environment.Similarly, the polynucleotides and polypeptides may occur in acomposition, such as a media formulations, solutions for introduction ofpolynucleotides or polypeptides, for example, into cells, compositionsor solutions for chemical or enzymatic reactions, for instance, whichare not naturally occurring compositions, and, therein remain isolatedpolynucleotides or polypeptides within the meaning of that term as it isemployed herein.

LIGATION refers to the process of forming phosphodiester bonds betweentwo or more polynucleotides, which most often are double stranded DNAs.Techniques for ligation are well known to the art and protocols forligation are described in standard laboratory manuals and references,such as, for instance, Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) and Maniatis et al., pg. 146, as cited below.

OLIGONUCLEOTIDE (S) refers to relatively short polynucleotides. Oftenthe term refers to single-stranded deoxyribonucleotides, but it canrefer as well to single- or double-stranded ribonucleotides, RNA:DNAhybrids and double-stranded DNAs, among others.

Oligonucleotides, such as single-stranded DNA probe oligonucleotides,often are synthesized by chemical methods, such as those implemented onautomated oligonucleotide synthesizers. However, oligonucleotides can bemade by a variety of other methods, including in vitro recombinantDNA-mediated techniques and by expression of DNAs in cells andorganisms.

Initially, chemically synthesized DNAs typically are obtained without a5′ phosphate. The 5′ ends of such oligonucleotides are not substratesfor phosphodiester bond formation by ligation reactions that employ DNAligases typically used to form recombinant DNA molecules. Where ligationof such oligonucleotides is desired, a phosphate can be added bystandard techniques, such as those that employ a kinase and ATP.

The 3′ end of a chemically synthesized oligonucleotide generally has afree hydroxyl group and, in the presence of a ligase, such as T4 DNAligase, readily will form a phosphodiester bond with a 5′ phosphate ofanother polynucleotide, such as another oligonucleotide. As is wellknown, this reaction can be prevented selectively, where desired, byremoving the 5′ phosphates of the other polynucleotide(s) prior toligation.

PLASMIDS generally are designated herein by a lower case p precededand/or followed by capital letters and/or numbers, in accordance withstandard naming conventions that are familiar to those of skill in theart. Starting plasmids disclosed herein are either commerciallyavailable, publicly available on an unrestricted basis, or can beconstructed from available plasmids by routine application of wellknown, published procedures. Many plasmids and other cloning andexpression vectors that can be used in accordance with the presentinvention are well known and readily available to those of skill in theart. Moreover, those of skill readily may construct any number of otherplasmids suitable for use in the invention. The properties, constructionand use of such plasmids, as well as other vectors, in the presentinvention will be readily apparent to those of skill from the presentdisclosure.

POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single- and double-stranded DNA, DNA that is a mixtureof single-and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, polynucleotide as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.

As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein.

It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

POLYPEPTIDES, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types.

It will be appreciated that polypeptides often contain amino acids otherthan the 20 amino acids commonly referred to as the 20 naturallyoccurring amino acids, and that many amino acids, including the terminalamino acids, may be modified in a given polypeptide, either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques which arewell known to the art. Even the common modifications that occurnaturally in polypeptides are too numerous to list exhaustively here,but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art. Among the known modificationswhich may be present in polypeptides of the present are, to name anillustrative few, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Analysis for protein modifications andnonprotein cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992).

It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of posttranslationevents, including natural processing event and events brought about byhuman manipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcell often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to express efficiently mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification may be presentin the same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

VARIANT(S) of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. Variants in this sense aredescribed below and elsewhere in the present disclosure in greaterdetail.

(1) A polynucleotide that differs in nucleotide sequence from another,reference polynucleotide. Generally, differences are limited so that thenucleotide sequences of the reference and the variant are closelysimilar overall and, in many regions, identical.

As noted below, changes in the nucleotide sequence of the variant may besilent. That is, they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed below.

(2) A polypeptide that differs in amino acid sequence from another,reference polypeptide. Generally, differences are limited so that thesequences of the reference and the variant are closely similar overalland, in many region, identical.

A variant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

RECEPTOR MOLECULE, as used herein, refers to molecules which bind orinteract specifically with hESF I, II and III polypeptides of thepresent invention, including not only classic receptors, which arepreferred, but also other molecules that specifically bind to orinteract with polypeptides of the invention (which also may be referredto as “binding molecules” and “interaction molecules,” respectively andas “hESF I, II and III binding molecules” and “hESF I, II and IIIinteraction molecules.” Binding between polypeptides of the inventionand such molecules, including receptor or binding or interactionmolecules may be exclusive to polypeptides of the invention, which isvery highly preferred, or it may be highly specific for polypeptides ofthe invention, which is highly preferred, or it may be highly specificto a group of proteins that includes polypeptides of the invention whichis preferred, or it may be specific to several groups of proteins atleast one of which includes polypeptides of the invention.

Receptors also may be non-naturally occurring, such as antibodies andantibody-derived reagents that bind to polypeptides of the invention.

DESCRIPTION OF THE INVENTION

The present invention relates to novel hESF I, II and III polypeptidesand polynucleotides, among other things, as described in greater detailbelow. In particular, the invention relates to polypeptides andpolynucleotides of novel human hESF I, II and III, which are related byamino acid sequence homology to the rat, prostatic steroid-bindingprotein. The invention relates especially to hESF I, II and III havingthe nucleotide and amino acid sequences set out in FIGS. 1, 2 and 3 (SEQID NO:1-6) and to the hESF I, II and III nucleotides and amino acidsequences of the human cDNAs in ATCC Deposit No. 97401, 97402 and 97403which is herein referred to as “the deposited clone” or as the “cDNA ofthe deposited clone.” It will be appreciated that the nucleotide andamino acid sequences set out in FIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and 6)were obtained by sequencing the cDNA of the deposited clone. Hence, thesequence of the deposited clone is controlling as to any discrepanciesbetween the two and any reference to the sequences of FIGS. 1, 2 and 3(SEQ ID NO:1, 3 and 5) include reference to the sequence of the humancDNA of the deposited claim.

Polynucleotides

In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode hESF I, II and IIIpolypeptides having the deduced amino acid sequences of FIGS. 1, 2 and 3(SEQ ID NO:2, 4 and 6).

Using the information provided herein, such as the polynucleotidesequences set out in FIGS. 1, 2 and 3 (SEQ ID NO:1, 3 and 5) apolynucleotide of the present invention encoding human hESF I, II andIII polypeptided may be obtained using standard cloning and screeningprocedures, such as those for cloning cDNAs using MRNA from cells of ahuman endometrial tumor as starting material. Illustrative of theinvention, the polynucleotide set out in FIG. 1 (SEQ ID NO:1) wasdiscovered in a cDNA library derived from cells of a human endometrialtumor. The polynucleotide of FIG. 2 (SEQ ID NO:3) was discovered in acDNA library derived from cyclohexamide treated CEM cells. Thepolynucleotide of FIG. 3 (SEQ ID NO:5) was discovered in a cDNA libraryderived from human endometrial tumor.

Human hESF I of the invention is structurally related to other proteinsof the Clara cell secretory protein family, as shown by the results ofsequencing the cDNA encoding human hESF I in the deposited clone. ThecDNA sequence thus obtained is set out in FIG. 1 (SEQ ID NO:1). Itcontains an open reading frame encoding a protein of about 90 amino acidresidues, wherein the initial 21 amino acid residues represent aputative leader sequence, with a deduced molecular weight of thefull-length protein of about 9.8 kDa. The protein exhibits greatesthomology to the rat prostatic steroid-binding protein, among knownproteins. The protein hESF I has about 46.067% identity and about 66.3%similarity with the amino acid sequence of the rat prostaticsteroid-binding protein.

Human hESP II contains an open reading frame encoding a protein of about90 amino acid residues, wherein the initial 21 amino acid residuesrepresent a putative leader sequence, with a deduced molecular weight ofthe full-length protein of about 9.9 kDa. The protein exhibits greatesthomology to the rat prostatic steroid-binding protein, among knownproteins. The protein hESF II has about 49.438% identity and about71.910% similarity with the amino acid sequence of rat prostaticsteroid-binding protein C2.

Human hESF III contains an open reading frame encoding a protein ofabout 95 amino acid residues, wherein the initial 21 amino acid residuesrepresent a putative leader sequence, with a deduced molecular weight ofthe full-length protein of about 8.10 kDa. The protein exhibits greatesthomology to rat, prostatic steroid-binding protein C3, among knownproteins. The protein hESF III has about 36.2% identity and about 64.9%similarity with the amino acid sequence of the rat prostaticsteroid-binding protein C3.

Polynucleotides of the present invention may be in the form of RNA, suchas mRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

The coding sequence which encodes the polypeptides may be identical tothe coding sequence of the polynucleotides shown in FIGS. 1, 2 and 3(SEQ ID NO:1, 3 and 5). It also may be a polynucleotide with a differentsequence, which, as a result of the redundancy (degeneracy) of thegenetic code, encodes the polypeptides of the human cDNA of FIGS. 1, 2and 3 (SEQ ID NO:1, 3 and 5).,

Polynucleotides of the present invention which encode the polypeptidesof FIGS. 1, 2 and 3 (SEQ ID NO:1, 3 and 5) may include, but are notlimited to the coding sequence for the mature polypeptide, by itself;the coding sequence for the mature polypeptide and additional codingsequences, such as those encoding a leader or secretory sequence, suchas a pre-, or pro- or prepro-protein sequence; the coding sequence ofthe mature polypeptide, with or without the aforementioned additionalcoding sequences, together with additional, non-coding sequences,including for example, but not limited to introns and non-coding 5′ and3′ sequences, such as the transcribed, non-translated sequences thatplay a role in transcription, mRNA processing—including splicing andpolyadenylation signals, for example—ribosome binding and stability ofmRNA; additional coding sequence which codes for additional amino acids,such as those which provide additional functionalities. Thus, forinstance, the polypeptide may be fused to a marker sequence, such as apeptide, which facilitates purification of the fused polypeptide. Incertain preferred embodiments of this aspect of the invention, themarker sequence is a hexa-histidine peptide, such as the tag provided inthe pQE vector (Qiagen, Inc., among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci., USA 86: 821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. The HA tag corresponds toan epitope derived of influenza hemagglutinin protein, which has beendescribed by Wilson et al., Cell 37: 767 (1984), for instance.

In accordance with the foregoing, the term “polynucleotide encoding apolypeptide” as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the present invention, particularlyhuman hESF I, II and III having the amino acid sequences set out inFIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and 6). The term encompassespolynucleotides that include a single continuous region or discontinuousregions encoding the polypeptide (for example, interrupted by introns)together with additional regions, that also may contain coding and/ornon-coding sequences.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptides having the deduced amino acid sequencesof FIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and 6). A variant of thepolynucleotide may be a naturally occurring variant such as a naturallyoccurring allelic variant, or it may be a variant that is not known tooccur naturally. Such non-naturally occurring variants of thepolynucleotide may be made by mutagenesis techniques, including thoseapplied to polynucleotides, cells or organisms.

Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions.

Among the particularly preferred embodiments of the invention in thisregard are polynucleotides encoding polypeptides having the amino acidsequence of hESF I, II and III set out in FIGS. 1, 2 and 3 (SEQ ID NO:2,4 and 6); variants, analogs, derivatives and fragments thereof, andfragments of the variants, analogs and derivatives.

Further particularly preferred in this regard are polynucleotidesencoding hESF I, II and III variants, analogs, derivatives andfragments, and variants, analogs and derivatives of the fragments, whichhave the amino acid sequence of the hESF I, II or III polypeptides ofFIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and 6) in which several, a few, 5 to10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted,deleted or added, in any combination. Especially preferred among theseare silent substitutions, additions and deletions, which do not alterthe properties and activities of the hESF I, II and III. Also especiallypreferred in this regard are conservative substitutions. Most. highlypreferred are polynucleotides encoding polypeptides having the aminoacid sequences of FIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and 6), withoutsubstitutions.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical to a polynucleotides encoding the hESF I, IIand III polypeptides having the amino acid sequences set out in FIGS. 1,2 and 3 (SEQ ID NO:2, 4 and 6), and polynucleotides which arecomplementary to such polynucleotides. Alternatively, most highlypreferred are polynucleotides that comprise a region that is at least80% identical to a polynucleotide encoding the hESF I, II or IIIpolypeptides of the cDNA of the deposited clone and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical to the same are particularly preferred, and among theseparticularly preferred polynucleotides, those with at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

Particularly preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological function or activity as the mature polypeptide encodedby the human cDNA of FIGS. 1, 2 and 3 (SEQ ID NO:1, 3 and 5).

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for cDNA and genomic DNA toisolate full-length cDNAs and genomic clones encoding hESF I, II or IIIand to isolate cDNA and genomic clones of other genes that have a highsequence similarity to the human hESF I, II or III genes. Such probesgenerally will comprise at least 15 bases. Preferably, such probes willhave at least 30 bases and may have at least 50 bases.

For example, the coding region of the hESF I, II and III genes may beisolated by screening using the known DNA sequence to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the present invention is then used toscreen a library of human cDNA, genomic DNA or MRNA to determine whichmembers of the library the probe hybridizes to.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease, as further discussed herein relatingto polynucleotide assays, inter alia.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may facilitateprotein trafficking, may prolong or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in situ, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Deposited Materials

A deposit containing a human hESF I, II and III cDNA has been depositedwith the American Type Culture Collection, as noted above. Also as notedabove, the cDNA deposit is referred to herein as “the deposited clone”or as “the cDNA of the deposited clone.”

The deposited clone was deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA,on Jan. 2, 1996 and assigned ATCC Deposit No. 97401, 97402 and 97403.

The deposited materials are pBluescript SK (−) plasmids (Stratagene, LaJolla, Calif.) containing the full length hESF I, II and III cDNA.

The deposit has been made under the terms of the Budapest Treaty on theinternational recognition of the deposit of micro-organisms for purposesof patent procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon, the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

The sequence of the polynucleotides contained in the deposited material,as well as the amino acid sequence of the polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

A license may be required to make, use or sell the deposited materials,and no such license is hereby granted.

Polypeptides

The present invention further relates to a human hESF I, II and IIIpolypeptide which has the deduced amino acid sequence of FIGS. 1, 2 and3 (SEQ ID NO:2, 4 and 6).

The invention also relates to fragments, analogs and derivatives ofthese polypeptides. The terms “fragment,” “derivative” and “analog” whenreferring to the polypeptide of FIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and 6)means a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog includes aproprotein which can be activated by cleavage of the proprotein portionto produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments it is a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIGS. 1, 2 and3 (SEQ ID NO:2, 4 and 6) may be (i) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) one in which one or more of the amino acidresidues includes a substituent group, or (iii) one in which the maturepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe mature polypeptide, such as a leader or secretory sequence or asequence which is employed for purification of the mature polypeptide ora proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 75% similarity (preferably at least 75% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

Fragments

Also among preferred embodiments of this aspect of the present inventionare polypeptides comprising fragments of hESF I, II and III, mostparticularly fragments of the hESF I, II and III having the amino acidsequence set out in FIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and 6), andfragments of variants and derivatives of the hESF I, II and III of FIGS.1, 2 and 3 (SEQ ID NO:2, 4 and 6).

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned hESF I, II and III polypeptides and variants orderivatives thereof.

Such fragments may be “free-standing,” i.e., not part of or fused toother amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of a hESF I, II orIII polypeptide of the present comprised within a precursor polypeptidedesigned for expression in a host and having heterologous pre andpro-polypeptide regions fused to the amino terminus of the hESF I, IIand III fragment and an additional region fused to the carboxyl terminusof the fragment. Therefore, fragments in one aspect of the meaningintended herein, refers to the portion or portions of a fusionpolypeptide or fusion protein derived from hESF I, II and III.

As representative examples of polypeptide fragments of the invention,there may be mentioned those which have from about 15 to about 139 aminoacids.

In this context about includes the particularly recited range and rangeslarger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes. Highly preferred in this regard arethe recited ranges plus or minus as many as 5 amino acids at either orat both extremes. Particularly highly preferred are the recited rangesplus or minus as many as 3 amino acids at either or at both the recitedextremes. Especially preferred are ranges plus or minus 1 amino acid ateither or at both extremes or the recited ranges with no additions ordeletions. Most highly preferred of all in this regard are fragmentsfrom about 15 to about 45 amino acids.

Among especially preferred fragments of the invention are truncationmutants of hESF I, II and III. Truncation mutants include hESF I, II andIII polypeptides having the amino acid sequence of FIGS. 1, 2 and 3 (SEQID NO:2, 4 and 6), or variants or derivatives thereof, except fordeletion of a continuous series of residues (that is, a continuousregion, part or portion) that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus or, asin double truncation mutants, deletion of two continuous series ofresidues, one including the amino terminus and one including thecarboxyl terminus. Fragments having the size ranges set out about alsoare preferred embodiments of truncation fragments, which are especiallypreferred among fragments generally.

Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of hESF I, II andIII. Preferred embodiments of the invention in this regard includefragments that comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet-forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of hESF I, II and III.

Certain preferred regions in these regards are set out in FIGS. 4, 5 and6 and include, but are not limited to, regions of the aforementionedtypes identified by analysis of the amino acid sequence set out in FIGS.1, 2 and 3 (SEQ ID NO:2, 4 and 6). As set out in FIGS. 4, 5 and 6 suchpreferred regions include Garnier-Robson alpha-regions, beta-regions,turn-regions and coil-regions, Chou-Fasman alpha-regions, beta-regionsand turn-regions, Kyte-Doolittle hydrophilic regions and hydrophilicregions, Eisenberg alpha and beta amphipathic regions, Karplus-Schulzflexible regions, Emini surface-forming regions and Jameson-Wolf highantigenic index regions.

Among highly preferred fragments in this regard are those that compriseregions of hESF I, II and III that combine several structural features,such as several of the features set out above. In this regard, theregions defined by the residues of FIGS. 1, 2 and 3 (SEQ ID NO:2, 4 and6), which all are characterized by amino acid compositions highlycharacteristic of turn-regions, hydrophilic regions, flexible-regions,surface-forming regions, and high antigenic index-regions, areespecially highly preferred regions. Such regions may be comprisedwithin a larger polypeptide or may be by themselves a preferred fragmentof the present invention, as discussed above. It will be appreciatedthat the term “about” as used in this paragraph has the meaning set outabove regarding fragments in general.

Further preferred regions are those that mediate activities of hESF I,II and III. Most highly preferred in this regard are fragments that havea chemical, biological or other activity of hESF I, II and III,including those with a similar activity or an improved activity, or witha decreased undesirable activity. Highly preferred in this regard arefragments that contain regions that are homologs in sequence, or inposition, or in both sequence and to active regions of relatedpolypeptides, such as the related polypeptides set out in FIGS. 4, 5 and6 (SEQ ID NO:2, 4 and 6) and which include rat prostaticspecific-binding proteins. Among particularly preferred fragments inthese regards are truncation mutants, as discussed above.

It will be appreciated that the invention also relates to, among others,polynucleotides encoding the aforementioned fragments, polynucleotidesthat hybridize to polynucleotides encoding the fragments, particularlythose that hybridize under stringent conditions, and polynucleotides,such as PCR primers, for amplifying polynucleotides that encode thefragments. In these regards, preferred polynucleotides are those thatcorrespondent to the preferred fragments, as discussed above.

Vectors, Host Cells, Expression

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. For instance,polynucleotides may be introduced into host cells using well knowntechniques of infection, transduction, transfection, transvection andtransformation. The polynucleotides may be introduced alone or withother polynucleotides. Such other polynucleotides may be introducedindependently, co-introduced or introduced joined to the polynucleotidesof the invention.

Thus, for instance, polynucleotides of the invention may be transfectedinto host cells with another, separate, polynucleotide encoding aselectable marker, using standard techniques for co-transfection andselection in, for instance, mammalian cells. In this case thepolynucleotides generally will be stably incorporated into the host cellgenome.

Alternatively, the polynucleotides may be joined to a vector containinga selectable marker for propagation in a host. The vector construct maybe introduced into host cells by the aforementioned techniques.Generally, a plasmid vector is introduced as DNA in a precipitate, suchas a calcium phosphate precipitate, or in a complex with a chargedlipid. Electroporation also may be used to introduce polynucleotidesinto a host. If the vector is a virus, it may be packaged in vitro orintroduced into a packaging cell and the packaged virus may betransduced into cells. A wide variety of techniques suitable for makingpolynucleotides and for introducing polynucleotides into cells inaccordance with this aspect of the invention are well known and routineto those of skill in the art. Such techniques are reviewed at length inSambrook et al. cited above, which is illustrative of the manylaboratory manuals that detail these techniques. In accordance with thisaspect of the invention the vector may be, for example, a plasmidvector, a single or double-stranded phage vector, a single ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells as polynucleotides, preferably DNA, by well known techniquesfor introducing DNA and RNA into cells. The vectors, in the case ofphage and viral vectors also may be and preferably are introduced intocells as packaged or encapsidated virus by well known techniques forinfection and transduction. Viral vectors may be replication competentor replication defective. In the latter case viral propagation generallywill occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression. Such specific expression may be inducibleexpression or expression only in certain types of cells or bothinducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

The engineered host cells can be cultured in conventional nutrientmedia, which may be modified as appropriate for, inter alia, activatingpromoters, selecting transformants or amplifying genes. Cultureconditions, such as temperature, pH and the like, previously used withthe host cell selected for expression generally will be suitable forexpression of polypeptides of the present invention as will be apparentto those of skill in the art.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids, all may be used for expression inaccordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques. In general, a DNA sequencefor expression is joined to an expression vector by cleaving the DNAsequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. Procedures for restriction and ligation that can be usedto this end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseskill, are set forth in great detail in Sambrook et al. cited elsewhereherein.

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name just a few of the well-known promoters. It will beunderstood that numerous promoters not mentioned are suitable for use inthis aspect of the invention are well known and readily may be employedby those of skill in the manner illustrated by the discussion and theexamples herein.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include a translationinitiating AUG at the beginning and a termination codon appropriatelypositioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as repressor binding sites and enhancers, amongothers.

Vectors for propagation and expression generally will include selectablemarkers. Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose. In this regard,the expression vectors preferably contain one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells. Preferred markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, and tetracycline or ampicillinresistance genes for culturing E. coli and other bacteria.

The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of agreat variety of expression constructs are well known, and those ofskill will be enabled by the present disclosure readily to select a hostfor expressing a polypeptides in accordance with this aspect of thepresent invention.

More particularly, the present invention also includes recombinantconstructs, such as expression constructs, comprising one or more of thesequences described above. The constructs comprise a vector, such as aplasmid or viral vector, into which such a sequence of the invention hasbeen inserted. The sequence may be inserted in a forward or reverseorientation. In certain preferred embodiments in this regard, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and there are many commercially available vectors suitable for use inthe present invention.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, PRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. These vectors are listed solelyby way of illustration of the many commercially available and well knownvectors that are available to those of skill in the art for use inaccordance with this aspect of the present invention. It will beappreciated that any other plasmid or vector suitable for, for example,introduction, maintenance, propagation or expression of a polynucleotideor polypeptide of the invention in a host may be used in this aspect ofthe invention.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase (“cat”) transcription unit,downstream of restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the cat gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable. Two such vectors are pKK232-8 and pCM7. Thus, promoters forexpression of polynucleotides of the present invention include not onlywell known and readily available promoters, but also promoters thatreadily may be obtained by the foregoing technique, using a reportergene.

Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacI and lacZ and promoters, the T3 and T7promoters, the gpt promoter, the lambda PR, PL promoters and the trppromoter. Among known eukaryotic promoters suitable in this regard arethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”), and metallothionein promoters,such as the mouse metallothionein-I promoter.

Selection of appropriate vectors and promoters for expression in a hostcell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

The present invention also relates to host cells containing theabove-described constructs discussed above. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host/cell can be a prokaryotic cell,such as a bacterial cell.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al. BASIC METHODS IN MOLECULARBIOLOGY, (1986).

Constructs in host cells can be used in a conventional manner to producethe gene product encoded by the recombinant sequence. Alternatively, thepolypeptides of the invention can be synthetically produced byconventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector. Among suitable promoters are those derived from the genesthat encode glycolytic enzymes such as 3-phosphoglycerate kinase(“PGK”), a-factor, acid phosphatase, and heat shock proteins, amongothers. Selectable markers include the ampicillin resistance gene of E.coli and the trp1 gene of S. cerevisiae.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Polynucleotides of the invention, encoding the heterologous structuralsequence of a polypeptide of the invention generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5′ to aribosome binding site. The ribosome binding site will be 5′ to the AUGthat initiates translation of the polypeptide to be expressed.Generally, there will be no other open reading frames that begin with aninitiation codon, usually AUG, and lie between the ribosome binding siteand the initiating AUG. Also, generally, there will be a translationstop codon at the end of the polypeptide and there will be apolyadenylation signal and a transcription termination signalappropriately disposed at the 3′ end of the transcribed region.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art.

Suitable prokaryotic hosts for propagation, maintenance or expression ofpolynucleotides and polypeptides in accordance with the inventioninclude Escherischia coli, Bacillus subtilis and Salmonella typhimurium.Various species of Pseudomonas, Streptomyces, and Staphylococcus aresuitable hosts in this regard. Moreover, many other hosts also known tothose of skill may be employed in this regard.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, where the selected promoteris inducible it is induced by appropriate means (e.g., temperature shiftor exposure to chemical inducer) and cells are cultured for anadditional period.

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can be employed for expression,as well. Examples of mammalian expression systems include the COS-7lines of monkey kidney fibroblast, described in Gluzman et al., Cell 23:175 (1981). Other cell lines capable of expressing a compatible vectorinclude for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHKcell lines.

Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking non-transcribedsequences that are necessary for expression. In certain preferredembodiments in this regard DNA sequences derived from the SV40 splicesites, and the SV40 polyadenylation sites are used for requirednon-transcribed genetic elements of these types.

The hESF I, II and III polypeptide can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Well known techniques for refolding protein may be employed toregenerate active conformation when the polypeptide is denatured duringisolation and or purification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

hESF I, II and III polynucleotides and polypeptides may be used inaccordance with the present invention for a variety of applications,particularly those that make use of the chemical and biologicalproperties hESF I, II and III. Additional applications relate todiagnosis and to treatment of disorders of cells, tissues and organisms.These aspects of the invention/are illustrated further by the followingdiscussion.

Polynucleotide Assays

This invention is also related to the use of the hESF I, II and IIIpolynucleotides to detect complementary polynucleotides such as, forexample, as a diagnostic reagent. Detection of a mutated form of hESF I,II and III associated with a dysfunction will provide a diagnostic toolthat can add or define a diagnosis of a disease or susceptibility to adisease which results from under-expression, over-expression or alteredexpression of hESF I, II and III, such as, for example, a susceptibilityto inherited asthma and endometrial cancer.

Individuals carrying mutations in the human hESF I, II and III gene maybe detected at the DNA level by a variety of techniques. Nucleic acidsfor diagnosis may be obtained from a patient's cells, such as fromblood, urine, saliva, tissue biopsy and autopsy material. The genomicDNA may be used directly for detection or may be amplified enzymaticallyby using PCR prior to analysis. PCR (Saiki et al., Nature, 324: 163-166(1986)). RNA or cDNA may also be used in the same ways. As an example,PCR primers complementary to the nucleic acid encoding hESF I, II andIII can be used to identify and analyze hESF I, II and III expressionand mutations. For example, deletions and insertions can be detected bya change in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled hESF I, II and III RNA or alternatively, radiolabeledhESF I, II and III antisense DNA sequences. Perfectly matched sequencescan be distinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230: 1242 (1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Chromosome Assays

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a hESF I, II and III gene.This can be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthe is used for in situ chromosome mapping using well known techniquesfor this purpose. Typically, in accordance with routine procedures forchromosome mapping, some trial and error may be necessary to identify agenomic probe that gives a good in situ hybridization signal.

In some cases, in addition, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60. For a review of this technique, see verma et al.,HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, NewYork (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,MENDELIAN INHERITANCE IN MAN, available on line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Polypeptide Assays

The present invention also relates to a diagnostic assays such asquantitative and diagnostic assays for detecting levels of hESF I, IIand III protein in cells and tissues, and biological fluids such, forexample, as blood and urine, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting over-expression or under-expression ofhESF I, II and III protein compared to normal control tissue samples maybe used to detect the presence of neoplasia, for example. Assaytechniques that can be used to determine levels of a protein, such as anhESF I, II and III protein of the present invention, in a sample derivedfrom a host are well-known to those of skill in the art. Such assaymethods include radioimmunoassays, competitive-binding assays, WesternBlot analysis and ELISA assays. Among these ELISAs frequently arepreferred. An ELISA assay initially comprises preparing an antibodyspecific to hESF I, II or III, preferably a monoclonal antibody. Inaddition a reporter antibody generally is prepared which binds to themonoclonal antibody. The reporter antibody is attached a detectablereagent such as radioactive, fluorescent or enzymatic reagent, forexample horseradish peroxidase enzyme.

To carry out an ELISA a sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein such as bovine serum albumin.Next, the monoclonal antibody is incubated in the dish during which timethe monoclonal antibodies attach to any hESF I, II or III proteinsattached to the polystyrene dish. Unbound monoclonal antibody is washedout with buffer. The reporter antibody linked to horseradish peroxidaseis placed in the dish resulting in binding of the reporter antibody toany monoclonal antibody bound to hESF I, II or III. Unattached reporterantibody is then washed out. Reagents for peroxidase activity, includinga colorimetric substrate are then added to the dish. Immobilizedperoxidase, linked to hESF I, II or III through the primary andsecondary antibodies, produces a colored reaction product. The amount ofcolor developed in a given time period indicates the amount of hESF I,II or III protein present in the sample. Quantitative results typicallyare obtained by reference to a standard curve.

A competition assay may be employed wherein antibodies specific to hESFI, II or III attached to a solid support and labeled hESF I, II or IIIand a sample derived from the host are passed over the solid support andthe amount of label detected attached to the solid support can becorrelated to a quantity of hESF I, II or III in the sample.

Antibodies

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Nature 256: 495-497 (1975), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies to immunogenic polypeptide products of thisinvention.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Thus, among others, the polynucleotides and polypeptides of the presentinvention may be employed to prevent and/or treat inflammation, asthma,rhinitis, cystic fibrosis, airway disease, prevent and/or treatneoplasia, atopy, inhibit phospholipase A₂, bind polychloratedbiphenyls, reduce foreign protein antigenicity, inhibit monocyte andneutrophil chemotaxis and phagocytosis, inhibit platelet aggregation,regulate eicosanoid levels in the human uterus, control the growth ofendometrial cells.

hESF I, II and III Binding Molecules and Assays

This invention also provides a method for identification of molecules,such as receptor molecules, that bind hESF I, II and III. Genes encodingproteins that bind hESF I, II and III, such as receptor proteins, can beidentified by numerous methods known to those of skill in the art, forexample, ligand panning and FACS sorting. Such methods are described inmany laboratory manuals such as, for instance, Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

For instance, expression cloning may be employed for this purpose. Tothis end polyadenylated RNA is prepared from a cell responsive to hESFI, II and III, a cDNA library is created from this RNA, the library isdivided into pools and the pools are transfected individually into cellsthat are not responsive to hESF I, II and III. The transfected cellsthen are exposed to labeled hESF I, II and III. (hESF I, II and III canbe labeled by a variety of well-known techniques including standardmethods of radio-iodination or inclusion of a recognition site for asite-specific protein kinase.) Following exposure, the cells are fixedand binding of cytostatin is determined. These procedures convenientlyare carried out on glass slides.

Pools are identified of cDNA that produced hESF I, II and III-bindingcells. Sub-pools are prepared from these positives, transfected intohost cells and screened as described above. Using an iterativesub-pooling and re-screening process, one or more single clones thatencode the putative binding molecule, such as a receptor molecule, canbe isolated.

Alternatively a labeled ligand can be photoaffinity linked to a cellextract, such as a membrane or a membrane extract, prepared from cellsthat express a molecule that it binds, such as a receptor molecule.Cross-linked material is resolved by polyacrylamide gel electrophoresis(“PAGE”) and exposed to X-ray film. The labeled complex containing theligand-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing can be used to design unique or degenerateoligonucleotide probes to screen cDNA libraries to identify genesencoding the putative receptor molecule.

Polypeptides of the invention also can be used to assess hESF I, II andIII binding capacity of hESF I, II and III binding molecules, such asreceptor molecules, in cells or in cell-free preparations.

Agonists and Antagonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose which enhance or block the action of hESF I, II and III on cells,such as its interaction with hESF I, II and III-binding molecules suchas receptor molecules. An agonist is a compound which increases thenatural biological functions of hESF I, II and III or which functions ina manner similar to hESF I, II and III, while antagonists decrease oreliminate such functions.

For example, a cellular compartment, such as a membrane or a preparationthereof, such as a membrane-preparation, may be prepared from a cellthat expresses a molecule that binds hESF I, II and III, such as amolecule of a signaling or regulatory pathway modulated by hESF I, IIand III. The preparation is incubated with labeled hESF I, II and III inthe absence or the presence of a candidate molecule which may be a hESFI, II and III agonist or antagonist. The ability of the candidatemolecule to bind the binding molecule is reflected in decreased bindingof the labeled ligand. Molecules which bind gratuitously, i.e., withoutinducing the effects of hESF I, II and III on binding the hESF I, II andIII binding molecule, are most likely to be good antagonists. Moleculesthat bind well and elicit effects that are the same as or closelyrelated to hESF I, II and III are agonists.

hESF I, II and III-like effects of potential agonists and antagonistsmay by measured, for instance, by determining activity of a secondmessenger system following interaction of the candidate molecule with acell or appropriate cell preparation, and comparing the effect with thatof hESF I, II and III or molecules that elicit the same effects as hESFI, II and III. Second messenger systems that may be useful in thisregard include but are not limited to AMP guanylate cyclase, ion channelor phosphoinositide hydrolysis second messenger systems.

Another example of an assay for hESF I, II and III antagonists is acompetitive assay that combines hESF I, II and III and a potentialantagonist with membrane-bound hESF I, II and III receptor molecules orrecombinant hESF I, II and III receptor molecules under appropriateconditions for a competitive inhibition assay. hESF I, II and III can belabeled, such as by radioactivity, such that the number of hESF I, IIand III molecules bound to a receptor molecule can be determinedaccurately to assess the effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducing hESF I,II and III-induced activities, thereby preventing the action of hESF I,II and III by excluding hESF I, II and III from binding.

Potential antagonists include a small molecule which binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such as receptor molecules, such thatnormal biological activity is prevented. Examples of small moleculesinclude but are not limited to small organic molecules, peptides orpeptide-like molecules.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed, for example, in—Okano, J. Neurochem. 56: 560 (1991);OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooneyet al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360(1991). The methods are based on binding of a polynucleotide to acomplementary DNA or RNA. For example, the 5′ coding portion of apolynucleotide that encodes the mature polypeptide of the presentinvention may be used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcriptionthereby preventing transcription and the production of hESF I, II andIII. The antisense RNA oligonucleotide hybridizes to the MRNA in vivoand blocks translation of the mRNA molecule into hESF I, II and IIIpolypeptide. The oligonucleotides described above can also be deliveredto cells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of hESF I, II and III.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The antagonists may be employed for instance to treat an inheritedsusceptibility to asthma.

Compositions

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or the agonists or antagonists.Thus, the polypeptides of the present invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration.

Kits

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Administration

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 μg/kg to about 1mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

Gene Therapy

The hESF I, II and III polynucleotides, polypeptides, agonists andantagonists that are polypeptides may be employed in accordance with thepresent invention by expression of such polypeptides in vivo, intreatment modalities often referred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo,and the engineered cells then can be provided to a patient to be treatedwith the polypeptide. For example, cells may be engineered ex vivo bythe use of a retroviral plasmid vector containing RNA encoding apolypeptide of the present invention. Such methods are well-known in theart and their use in the present invention will be apparent from theteachings herein.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by procedures known in the art. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct then may be isolated and introduced intoa packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors herein abovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

Such vectors well include one or more promoters for expressing thepolypeptide. Suitable promoters which may be employed include, but arenot limited to, the retroviral LTR; the SV40 promoter; and the humancytomegalovirus (CMV) promoter described in Miller et al., Biotechniques7: 980-990 (1989), or any other promoter (e.g., cellular promoters suchas eukaryotic cellular promoters including, but not limited to, thehistone, RNA polymerase III, and β-actin promoters). Other viralpromoters which may be employed include, but are not limited to,adenovirus promoters, thymidine kinase (TK) promoters, and B19parvovirus promoters. The selection of a suitable promoter will beapparent to those skilled in the art from the teachings containedherein.

The nucleic acid sequence encoding the polypeptide of the presentinvention will be placed under the control of a suitable promoter.Suitable promoters which may be employed include, but are not limitedto, adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, Y-2,Y-AM, PA12, T19-14X, VT-19-17-H2, YCRE, YCRIP, GP+E−86, GP+envAm12, andDAN cell lines as described in Miller, A., Human Gene Therapy 1: 5-14(1990). The vector may be transduced into the packaging cells throughany means known in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO4 precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line will generate infectious retroviral vectorparticles, which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

EXAMPLES

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplification's, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), herein referred to as “Sambrook.”

All parts or amounts set out in the following examples are by weight,unless otherwise specified.

Unless otherwise stated size separation of fragments in the examplesbelow was carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis (“PAGE”) in Sambrook and numerousother references such as, for instance, by Goeddel et al., Nucleic AcidsRes. 8: 4057 (1980).

Unless described otherwise, ligations were accomplished using standardbuffers, incubation temperatures and times, approximately equimolaramounts of the DNA fragments to be ligated and approximately 10 units ofT4 DNA ligase (“ligase”) per 0.5 μg of DNA.

Example 1

Expression and Purification of Human hESF I, II and III Using Bacteria

The DNA sequence encoding human hESF I, II or III in the depositedpolynucleotide was amplified using PCR oligonucleotide primers specificto the amino acid carboxyl terminal sequence of the human hESF I, II orIII protein and to vector sequences 3′ to the gene. Additionalnucleotides containing restriction sites to facilitate cloning wereadded to the 5′ and 3′ sequences respectively.

The 5′ oligonucleotide primer had the sequence

-   -   for hESF I: 5′ CGCGCATGCTTGTCTGCCCAGCTG 3′ (SEQ ID NO:7)        containing the underlined SphI restriction site, which encodes a        start AUG, followed by 15 nucleotides of the human hESF I coding        sequence set out in FIG. 1 (SEQ ID NO:1), beginning with the        first base of the codon for amino acid 22 (leucine).    -   for hESF II: 5′ CGCCCATGGAGTTCTGCCCAGCTC 3′ (SEQ ID NO:8)        containing the underlined NcoI restriction site, which encodes a        start ATG, followed by 16 nucleotides of the human hESF II        coding sequence set out in FIG. 2 (SEQ ID NO:3), beginning with        the first base of the codon for amino acid 22.    -   for hESF III: 5′ CGC GCA TGC ACT GCT ATG CAG ATT 3′ (SEQ ID        NO:9) containing the underlined SphI restriction site, which        encodes a start ATG, followed by 16 nucleotides of the human        hESF III coding sequence set out in FIG. 3 (SEQ ID NO:5).

The 3′ Primer has the Sequence

-   -   for hESF I 5′ CGCAAGCTTCATTTTACATGTCA 3′ (SEQ ID NO:10)        containing the underlined Hind III restriction site followed by        15 nucleotides complementary to 15 nucleotides of the hESF I        non-coding sequence set out in FIG. 1 (SEQ ID NO:1), including        the stop codon.    -   for hESF II 5′ CGCAAGCTTAGTTTTTACATGTCA 3′ (SEQ ID NO:11)        containing the underlined Hind III restriction site followed by        15 nucleotides complementary to the last 15 nucleotides of the        hESF II non-coding sequence set out in FIG. 2 (SEQ ID NO:3),        including the stop codon.    -   for hESF III 5′ CGC AAG CTT ACG CCT TGG GTA AAG TTA 3′ (SEQ ID        NO:12) containing the underlined HindIII restriction site        followed by 18 nucleotides complementary to hESF III non-coding        sequence set out in FIG. 3 (SEQ ID NO:5), including the stop        codon.

The restrictions sites were convenient to restriction enzyme sites inthe bacterial expression vectors pQE-60 (hESF I and II), (Qiagen, Inc.)which were used for bacterial expression in these examples. (Qiagen,Inc. Chatsworth, Calif.). pQE-60 encodes ampicillin antibioticresistance (“Ampr”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a6-His tag and restriction enzyme sites.

The amplified human hESF I, II and III DNA and the vector pQE-60 bothwere digested with Sph I and Hind III (hESF I), Nco I and HindIII (hESFII) and SphI and HindIII (hESF III) and the digested DNAs then wereligated together. Insertion of the hESF I DNA into the restricted vectorplaced the respective coding regions downstream of and operably linkedto the vector's IPTG-inducible promoter and in-frame with an initiatingAUG appropriately positioned for translation of hESF I, II and III.

The ligation mixture was transformed into competent E. coli cells usingstandard procedures. Such procedures are described in Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kanr”), wasused in carrying out the illustrative example described here. Thisstrain, which is only one of many that are suitable for expressing hESFI, II and III is available commercially from Qiagen.

Transformants were identified by their ability to grow on LB plates inthe presence of ampicillin. Plasmid DNA was isolated from resistantcolonies and the identity of the cloned DNA was confirmed by restrictionanalysis.

Clones containing the desired constructs were grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml).

The O/N culture was used to inoculate a large culture, at a dilution ofapproximately 1:100 to 1:250. The cells were grown to an optical densityat 600nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) was then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently were incubated further for 3 to 4 hours. Cells then wereharvested by centrifugation and disrupted, by standard methods.Inclusion bodies were purified from the disrupted cells using routinecollection techniques, and protein was solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein was passed over a PD-10 column in 2× phosphate buffered saline(“PBS”), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein was purified by a further step ofchromatography to remove endotoxin. Then, it was sterile filtered. Thesterile filtered protein preparation was stored in 2× PBS at aconcentration of 95 micrograms per mL.

Analysis of the preparation by standard methods of polyacrylamide gelelectrophoresis revealed that the preparation contained about 95%monomer hESF I, II and III having the expected molecular weight.

Example 2

Cloning and Expression of Human hESF I, II and III in a BaculovirusExpression System

The cDNA sequence encoding the full length human hESF I, II and IIIprotein, in the deposited clone is amplified using PCR oligonucleotideprimers corresponding to the 5′ and 3′ sequences of the gene:

-   -   for hESF I the 5′ primer has the sequence 5′ CCCGGATCC        GCCATCATGAGGCTGTCAGTGTGTCT 3′ (SEQ ID NO:13) containing the        BamHI restriction enzyme site (bold) followed by a kozak        sequence (GCC ATC) and 20 bases of the sequence of hESF I of        FIG. 1 (SEQ ID NO:1);    -   for hESF II the 5′ primer has the sequence 5′ CGC GGA TCC GCC        ATC ATG AAG CTG TCG GTG 3′ (SEQ ID NO:14) containing the BamHI        restriction enzyme site (bold) followed by 15 bases of the        sequence of hESF II of FIG. 2 (SEQ ID NO:3);    -   for hESF III the 5′ primer has the sequence 5′ CGC GGA TCC GCC        ATC ATG AAG CTG CTG ATG GTC 3′ (SEQ ID NO:15) containing the        BamHI restriction enzyme site (bold) followed by 15 bases of the        sequence of hESF III of FIG. 3 (SEQ ID NO:5).

Inserted into an expression vector, as described below, the 5′ end ofthe amplified fragment encoding human hESF I, II or III provides anefficient signal peptide. An efficient signal for initiation oftranslation in eukaryotic cells, as described by Kozak, M., J. Mol.Biol. 196: 947-950 (1987) is appropriately located in the vector portionof the construct.

For hESF I the 3′ primer has the sequence 5′ CCCGGTACCTTTTTTTTTTTTTTTTTT3′ (SEQ ID NO:16) containing the underlined Asp718 restriction sitefollowed by 18 nucleotides complementary to the poly A tail;

for hESF II the 3′ primer has the sequence 5′ CGCGGTACCACGCCTTGGGTAAAGTTA 3′ (SEQ ID NO:17) containing the underlinedAsp718 restriction followed by nucleotides complementary to 15nucleotides of the hESF II non-coding sequence set out in FIG. 2 (SEQ IDNO:3), including the stop codon;

-   -   for hESF III the 3′ primer has the sequence 5′ CGC GGT ACC ACG        CCT TGG GTA AAG TTA 3′ (SEQ ID NO:18) containing the underlined        Asp718 restriction followed by nucleotides complementary to 18        nucleotides of the hESF III non-coding sequence set out in FIG.        3 (SEQ ID NO:5), including the stop codon.

The amplified fragments are isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragments then are digested with the respective restrictionenzymes and again are purified on a 1% agarose gel. This fragments aredesignated herein F2.

The vector pRG1 is used to express the hESF I, II or III protein in thebaculovirus expression system, using standard methods, such as thosedescribed in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORSAND INSECT CELL CULTURE PROCEDURES, Texas Agricultural ExperimentalStation Bulletin No. 1555 (1987). This expression vector contains thestrong polyhedrin promoter of the Autographa californica nuclearpolyhedrosis virus (AcMNPV) followed by convenient restriction sites.The polyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For an easy selection of recombinant virusthe beta-galactosidase gene from E.coli is inserted in the sameorientation as the polyhedrin promoter and is followed by thepolyadenylation signal of the polyhedrin gene. The polyhedrin sequencesare flanked at both sides by viral sequences for cell-mediatedhomologous recombination with wild-type viral DNA to generate viablevirus that express the cloned polynucleotide.

Many other baculovirus vectors could be used in place of pA2, such aspAc373, pVL941 and pAcIM1 provided, as those of skill readily willappreciate, that construction provides appropriately located signals fortranscription, translation, trafficking and the like. Such vectors aredescribed in Luckow et al., Virology 170: 31-39, among others.

The plasmid is digested with the respective restriction enzymes and thenis dephosphorylated using calf intestinal phosphatase, using routineprocedures known in the art. The DNA is then isolated from a 1% agarosegel using a commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Calif.). This vector DNA is designated herein “V2”.

Fragments F2 and the dephosphorylated plasmid V2 are ligated togetherwith T4 DNA ligase. E. coli HB101 cells are transformed with ligationmix and spread on culture plates. Bacteria are identified that containthe plasmid with the human hESF I, II or III gene by digesting DNA fromindividual colonies using the respective restriction enzymes and thenanalyzing the digestion product by gel electrophoresis. The sequence ofthe cloned fragment is confirmed by DNA sequencing. This plasmid isdesignated herein pBachESF I, II or III.

5 μg of the plasmid-pBachESF I, II or III is co-transfected with 1.0 μgof a commercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmidpBachESF I, II or III are mixed in a sterile well of a microtiter platecontaining 50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10).

Four days after serial dilution, the virus is added to the cells. Afterappropriate incubation, blue stained plaques are picked with the tip ofan Eppendorf pipette. The agar containing the recombinant viruses isthen resuspended in an Eppendorf tube containing 200 μl of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. A clonecontaining properly inserted hESF I, II or III is identified by DNAanalysis including restriction mapping and sequencing. This isdesignated herein as V-hESF I, II or III.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FES. The cells are infected with the recombinantbaculovirus V-hESF I, II or III at a multiplicity of infection (“MOI”)of about 2. Six hours later the medium is removed and is replaced withSF900 II medium minus methionine and cysteine (available from LifeTechnologies Inc., Gaithersburg). 42 hours later, 5 μCi of35S-methionine and 5 μCi 35S cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

Example 3

Expression of hESF I, II and III in COS Cells

The expression plasmid, hESF I, II and III HA, is made by cloning a cDNAencoding hESF I, II and III into the expression vector pcDNAI/Amp (whichcan be obtained from Invitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcell; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron, and a polyadenylation signal arranged so that a cDNAconveniently can be placed under expression control of the CMV promoterand operably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker.

A DNA fragment encoding the entire hESF I, II and III precursor and a HAtag fused in frame to its 3′ end is cloned into the polylinker region ofthe vector so that recombinant protein expression is directed by the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein described by Wilson et al., Cell 37: 767(1984). The fusion of the HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is as follows:

The hESF I, II and III cDNA of the deposit clone is amplified usingprimers that contained convenient restriction sites, much as describedabove regarding the construction of expression vectors for expression ofhESF I, II and III in E. coli and S. fugiperda.

To facilitate detection, purification and characterization of theexpressed hESF I, II and III, one of the primers contains aheamaglutinin tag (“HA tag”) as described above.

Suitable primers include that following, which are used in this example:

The 5′ primer, containing the underlined BamHI site, an AUG start codonand has the following sequence. 5′ CGC GGA TCC ACC ATG GTC TCG CTG GCCCTT 3′ (SEQ ID NO:19) (ESFI); 5′ CGC GGA TCC ACC ATG AAG CTG TCG GTG TGT3′ (SEQ ID NO:20) (ESFII); 5′ CGC GGA TCC ACC ATG AAG CTG CTG ATG GTC 3′(SEQ ID NO:21) (ESFIII).

The 3′ primer, containing the underlined XbaI site, stop codon, HA tagand 15 bp of 3′ coding sequence (at the 3′ end) has the followingsequence:

5′ CGC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CAC ACC ACA TTTTTT 3′  (SEQ ID NO:22) (ESFI); 5′ CGC TCT AGA TCA AGC GTA GTC TGG GACGTC GTA TGG GTA CAC ACT ACA TTT CTT 3′ (SEQ ID NO:23) (ESFII); 5′ CGCTCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA ATT ACT CTT CAT ATT 3′(SEQ ID NO:24) (ESFIII).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith and then ligated. The ligation mixture is transformed into E. colistrain SURE (available from Stratagene Cloning Systems, 11099 NorthTorrey Pines Road, La Jolla, Calif. 92037) the transformed culture isplated on ampicillin media plates which then are incubated to allowgrowth of ampicillin resistant colonies. Plasmid DNA is isolated fromresistant colonies and examined by restriction analysis and gel sizingfor the presence of the hESF I, II and III-encoding fragment.

For expression of recombinant hESF I, II and III, COS cells aretransfected with an expression vector, as described above, usingDEAE-DEXTRAN, as described, for instance, in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, Cold Spring Laboratory Press, Cold SpringHarbor, N.Y. (1989). Cells are incubated under conditions for expressionof hESF I, II and III by the vector.

Expression of the hESF I, II and III HA fusion protein is detected byradiolabelling and immunoprecipitation, using methods described in, forexample Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing 35S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE gels and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 4

Tissue Distribution of hESF I, II and III Expression

Northern blot analysis is carried out to examine the levels ofexpression of hESF I, II and III in human tissues, using methodsdescribed by, among others, Sambrook et al, cited above. Total cellularRNA samples are isolated With RNAzol™ B system (Biotecx Laboratories,Inc. 6023 South Loop East, Houston, Tex. 77033).

About 10 μg of Total RNA is isolated from tissue samples. The RNA issize resolved by electrophoresis through a 1% agarose gel under stronglydenaturing conditions. RNA is blotted from the gel onto a nylon filter,and the filter then is prepared for hybridization to a detectablylabeled polynucleotide probe.

As a probe to detect mRNA that encodes hESF I, II and III, the antisensestrand of the coding region of the cDNA insert in the deposited clone islabeled to a high specific activity. The cDNA is labeled by primerextension, using the Prime-It kit, available from Stratagene. Thereaction is carried out using 50 ng of the cDNA, following the standardreaction protocol as recommended by the supplier. The labeledpolynucleotide is purified away from other labeled reaction componentsby column chromatography using a Select-G-50 column, obtained from5-Prime-3-Prime, Inc. of 5603 Arapahoe Road, Boulder, Colo. 80303.

The labeled probe is hybridized to the filter, at a concentration of1,000,000 cpm/ml, in a small volume of 7% SDS, 0.5 M NaPO4, pH 7.4 at65° C., overnight.

Thereafter the probe solution is drained and the filter is washed twiceat room temperature and twice at 60° C. with 0.5×SSC, 0.1% SDS. Thefilter then is dried and exposed to film at −70° C. overnight with anintensifying screen.

Example 5

Gene Therapeutic Expression of Human hESF I, II and III

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature overnight. After 24 hours at room temperature, the flask isinverted—the chunks of tissue remain fixed to the bottom of theflask—and fresh media is added (e.g., Ham's F12 media, with 10% FBS,penicillin and streptomycin). The tissue is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerges. The monolayer istrypsinized and scaled into larger flasks.

A vector for gene therapy is digested with restriction enzymes forcloning a fragment to be expressed. The digested vector is treated withcalf intestinal phosphatase to prevent self-ligation. Thedephosphorylated, linear vector is fractionated on an agarose gel andpurified.

hESF I, II and III cDNA capable of expressing active hESF I, II and III,is isolated. The ends of the fragment are modified, if necessary, forcloning into the vector. For instance, 5″ overhanging may be treatedwith DNA polymerase to create blunt ends. 3′ overhanging ends may beremoved using S1 nuclease. Linkers may be ligated to blunt ends with T4DNA ligase.

Equal quantities of the Moloney murine leukemia virus linear backboneand the hESF I, II or III fragment are mixed together and joined usingT4 DNA ligase. The ligation mixture is used to transform E. Coli and thebacteria are then plated onto agar-containing kanamycin. Kanamycinphenotype and restriction analysis confirm that the vector has theproperly inserted gene.

Packaging cells are grown in tissue culture to confluent density inDulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS),penicillin and streptomycin. The vector containing the hESF I, II or IIIgene is introduced into the packaging cells by standard techniques.Infectious viral particles containing the hESF I, II or III gene arecollected from the packaging cells, which now are called producer cells.

Fresh media is added to the producer cells, and after an appropriateincubation period media is harvested from the plates of confluentproducer cells. The media, containing the infectious viral particles, isfiltered through a Millipore filter to remove detached producer cells.The filtered media then is used to infect fibroblast cells. Media isremoved from a sub-confluent plate of fibroblasts and quickly replacedwith the filtered media. Polybrene (Aldrich) may be included in themedia to facilitate transduction. After appropriate incubation, themedia is removed and replaced with fresh media. If the titer of virus ishigh, then virtually all fibroblasts will be infected and no selectionis required. If the titer is low, then it is necessary to use aretroviral vector that has a selectable marker, such as neo or his, toselect out transduced cells for expansion.

Engineered fibroblasts then may be injected into rats, either alone orafter having been grown to confluence on microcarrier beads, such ascytodex 3 beads. The injected fibroblasts produce hESF I, II or IIIproduct, and the biological actions of the protein are conveyed to thehost.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

1. An isolated antibody or portion thereof that specifically binds to aprotein selected from the group consisting of: (a) a protein whosesequence consists of amino acid residues−21 to 69 of SEQ ID NO: 4; (b) aprotein whose sequence consists of amino acid residues 1 to 69 of SEQ IDNO: 4; (c) a protein whose sequence consists of an antigenic fragment ofthe amino acid sequence of SEQ ID NO:4; (d) a protein consisting of afragment of SEQ ID NO:4, wherein said fragment comprises at least 30contiguous amino acid residues of SEQ ID NO:4; and (e) a proteinconsisting of a fragment of SEQ ID NO:4, wherein said fragment comprisesat least 50 contiguous amino acid residues of SEQ ID NO:4.
 2. Theantibody or portion thereof of claim 1 that specifically binds protein(a).
 3. The antibody or portion thereof of claim 1 that specificallybinds protein (b).
 4. The antibody or portion thereof of claim 1 thatspecifically binds protein (c).
 5. The antibody or portion thereof ofclaim 1 that specifically binds protein (d).
 6. The antibody or portionthereof of claim 1 that specifically binds protein (e).
 7. The antibodyor portion thereof of claim 1 wherein said protein specifically bound bysaid antibody or portion thereof is glycosylated.
 8. The antibody orportion thereof of claim 1 which is a monoclonal antibody.
 9. Theantibody or portion thereof of claim 1 which is a polyclonal antibody.10. The antibody or portion thereof of claim 1 which is a chimericantibody.
 11. The antibody or portion thereof of claim 1 which is ahumanized antibody.
 12. The antibody or portion thereof of claim 1 whichis a human antibody.
 13. The antibody or portion thereof of claim 1which is a single chain antibody.
 14. The antibody or portion thereof ofclaim 1 which is a Fab fragment.
 15. A composition comprising theantibody or portion thereof of claim 1 and a carrier.
 16. Thecomposition of claim 15, wherein the antibody or portion thereof is amonoclonal antibody.
 17. The composition of claim 15, wherein theantibody or portion thereof is a chimeric antibody.
 18. The compositionof claim 15, wherein the antibody or portion thereof is a humanizedantibody.
 19. The composition of claim 15, wherein the antibody orportion thereof is a human antibody.
 20. The composition of claim 15,wherein the antibody or portion thereof is a single chain antibody. 21.The composition of claim 15, wherein the antibody or portion thereof isa Fab fragment.
 22. An isolated cell that produces the antibody ofclaim
 1. 23. A hybridoma that produces the antibody of claim
 1. 24. Ahybridoma that produces the antibody of claim
 8. 25. A method ofdetecting hESF II protein in a biological sample comprising: (a)contacting the biological sample with the antibody or portion thereof ofclaim 1; and (b) detecting the hESF II protein in the biological sampleby its binding to the antibody or portion thereof.
 26. The method ofclaim 25 wherein the antibody is a monoclonal antibody.
 27. The methodof claim 25 wherein the antibody is a polyclonal antibody.
 28. Themethod of claim 25 wherein the antibody is a chimeric antibody.
 29. Themethod of claim 25 wherein the antibody is a humanized antibody.
 30. Themethod of claim 25 wherein the antibody is a human antibody.
 31. Themethod of claim 25 wherein the antibody is a single chain antibody. 32.An isolated antibody or portion thereof produced by immunizing an animalwith a protein selected from the group consisting of: (a) a proteinwhose sequence comprises amino acid residues−21 to 69 of SEQ ID NO: 4;(b) a protein whose sequence comprises amino acid residues 1 to 69 ofSEQ ID NO: 4; (c) a protein whose sequence comprises an antigenicfragment of the amino acid sequence of SEQ ID NO:4; (d) a protein whosesequence comprises at least 30 contiguous amino acid residues of SEQ IDNO:4; and (e) a protein whose sequence comprises at least 50 contiguousamino acid residues of SEQ ID NO:4, wherein said antibody or portionthereof specifically binds to the amino acid sequence of SEQ ID NO:4.33. A The antibody or portion thereof of claim 32 produced by immunizingan animal with protein (a).
 34. The antibody or portion thereof of claim32 produced by immunizing an animal with protein (b).
 35. The antibodyor portion thereof of claim 32 produced by immunizing an animal withprotein (c).
 36. The antibody or portion thereof of claim 32 produced byimmunizing an animal with protein (d).
 37. The antibody or portionthereof of claim 32 produced by immunizing an animal with protein (e).38. The antibody or portion thereof of claim 32 wherein said proteinspecifically bound by said antibody or portion thereof is glycosylated.39. The antibody or portion thereof of claim 32 which is a monoclonalantibody.
 40. The antibody or portion thereof of claim 32 which is apolyclonal antibody.
 41. The antibody or portion thereof of claim 32which is a chimeric antibody.
 42. The antibody or portion thereof ofclaim 32 which is a humanized antibody.
 43. The antibody or portionthereof of claim 32 which is a human antibody.
 44. The antibody orportion thereof of claim 32 which is a single chain antibody.
 45. Theantibody or portion thereof of claim 32 which is a Fab fragment.
 46. Acomposition comprising the antibody or portion thereof of claim 32 and acarrier.
 47. The composition of claim 46, wherein the antibody orportion thereof is a monoclonal antibody.
 48. The composition of claim46, wherein the antibody or portion thereof is a chimeric antibody. 49.The composition of claim 46, wherein the antibody or portion thereof isa humanized antibody.
 50. The composition of claim 46, wherein theantibody or portion thereof is a human antibody.
 51. The composition ofclaim 46, wherein the antibody or portion thereof is a single chainantibody.
 52. The composition of claim 46, wherein the antibody orportion thereof is a Fab fragment.
 53. An isolated cell that producesthe antibody of claim
 32. 54. A hybridoma that produces the antibody ofclaim
 32. 55. A hybridoma that produces the antibody of claim
 39. 56. Amethod of detecting hESF II protein in a biological sample comprising:(a) contacting the biological sample with the antibody or portionthereof of claim 32; and (b) detecting the hESF II protein in thebiological sample by its binding to the antibody or portion thereof. 57.The method of claim 56 wherein the antibody is a monoclonal antibody.58. The method of claim 56 wherein the antibody is a polyclonalantibody.
 59. The method of claim 56 wherein the antibody is a chimericantibody.
 60. The method of claim 56 wherein the antibody is a humanizedantibody.
 61. The method of claim 56 wherein the antibody is a humanantibody.
 62. The method of claim 56 wherein the antibody is a singlechain antibody.
 63. The method of claim 56 wherein the antibody orportion thereof is a Fab fragment.
 64. An isolated antibody or portionthereof that specifically binds to a protein selected from the groupconsisting of: (a) a protein whose sequence consists of the amino acidsequence of the full-length polypeptide encoded by the cDNA contained inATCC Deposit Number 97402; (b) a protein whose sequence consists of theamino acid sequence of the mature polypeptide encoded by the cDNAcontained in ATCC Deposit Number 97402; (c) a protein whose sequenceconsists of an antigenic fragment of the polypeptide encoded by the cDNAcontained in ATCC Deposit Number 97402; (d) a protein consisting of afragment of the polypeptide encoded by the cDNA contained in ATCCDeposit Number 97402, wherein said fragment comprises at least 30contiguous amino acid residues of the polypeptide encoded by the cDNAcontained in ATCC Deposit Number 97402; and (e) a protein consisting ofa fragment of the polypeptide encoded by the cDNA contained in ATCCDeposit Number 97402, wherein said fragment comprises at least 50contiguous amino acid residues of the polypeptide encoded by the cDNAcontained in ATCC Deposit Number
 97402. 65. The antibody or portionthereof of claim 64 that specifically binds protein (a).
 66. Theantibody or portion thereof of claim 64 that specifically binds protein(b).
 67. The antibody or portion thereof of claim 64 that specificallybinds protein (c).
 68. The antibody or portion thereof of claim 64 thatspecifically binds protein (d).
 69. The antibody or portion thereof ofclaim 64 that specifically binds protein (e).
 70. The antibody orportion thereof of claim 64 wherein said protein specifically bound bysaid antibody or portion thereof is glycosylated.
 71. The antibody orportion thereof of claim 64 which is a monoclonal antibody.
 72. Theantibody or portion thereof of claim 64 which is a polyclonal antibody.73. The antibody or portion thereof of claim 64 which is a chimericantibody.
 74. The antibody or portion thereof of claim 64 which is ahumanized antibody.
 75. The antibody or portion thereof of claim 64which is a human antibody.
 76. The antibody or portion thereof of claim64 which is a single chain antibody.
 77. The antibody or portion thereofof claim 64 which is a Fab fragment.
 78. A composition comprising theantibody or portion thereof of claim 64 and a carrier.
 79. Thecomposition of claim 78 wherein the antibody or portion thereof is amonoclonal antibody.
 80. The composition of claim 78 wherein theantibody or portion thereof is a chimeric antibody.
 81. The compositionof claim 78 wherein the antibody or portion thereof is a humanizedantibody.
 82. The composition of claim 78 wherein the antibody orportion thereof is a human antibody.
 83. The composition of claim 78wherein the antibody or portion thereof is a single chain antibody. 84.The composition of claim 78 wherein the antibody or portion thereof is aFab fragment.
 85. An isolated cell that produces the antibody of claim64.
 86. A hybridoma that produces the antibody of claim
 64. 87. Ahybridoma that produces the antibody of claim
 71. 88. A method ofdetecting hESF II protein in a biological sample comprising: (a)contacting the biological sample with the antibody or portion thereof ofclaim 64; and (b) detecting the hESF II protein in the biological sampleby its binding to the antibody or portion thereof.
 89. The method ofclaim 88 wherein the antibody is a monoclonal antibody.
 90. The methodof claim 88 wherein the antibody is a polyclonal antibody.
 91. Themethod of claim 88 wherein the antibody is a chimeric antibody.
 92. Themethod of claim 88 wherein the antibody is a humanized antibody.
 93. Themethod of claim 88 wherein the antibody is a human antibody.
 94. Themethod of claim 88 wherein the antibody is a single chain antibody. 95.An isolated antibody or portion thereof produced by immunizing an animalwith a protein selected from the group consisting of: (a) a proteinwhose sequence comprises the amino acid sequence of the full-lengthpolypeptide encoded by the cDNA contained in ATCC Deposit Number 97402;(b) a protein whose sequence comprises the amino acid sequence of themature polypeptide encoded by the cDNA contained in ATCC Deposit Number97402; (c) a protein whose sequence comprises an antigenic fragment ofthe polypeptide encoded by the cDNA contained in ATCC Deposit Number97402; (d) a protein whose sequence comprises at least 30 contiguousamino acid residues of the polypeptide encoded by the cDNA contained inATCC Deposit Number 97402; and, (e) a protein whose sequence comprisesat least 50 contiguous amino acid residues of the polypeptide encoded bythe cDNA contained in ATCC Deposit Number 97402; wherein said antibodyor portion thereof specifically binds to the polypeptide encoded by thecDNA contained in ATCC Deposit Number
 97402. 96. The antibody or portionthereof of claim 95 produced by immunizing an animal with protein (a).97. The antibody or portion thereof of claim 95 produced by immunizingan animal with protein (b).
 98. The antibody or portion thereof of claim95 produced by immunizing an animal with protein (c).
 99. The antibodyor portion thereof of claim 95 produced by immunizing an animal withprotein (d).
 100. The antibody or portion thereof of claim 95 producedby immunizing an animal with protein (e).
 101. The antibody or portionthereof of claim 95 wherein said protein specifically bound by saidantibody or portion thereof is glycosylated.
 102. The antibody orportion thereof of claim 95 which is a monoclonal antibody.
 103. Theantibody or portion thereof of claim 95 which is a polyclonal antibody.104. The antibody or portion thereof of claim 95 which is a chimericantibody.
 105. The antibody or portion thereof of claim 95 which is ahumanized antibody.
 106. The antibody or portion thereof of claim 95which is a human antibody.
 107. The antibody or portion thereof of claim95 which is a single chain antibody.
 108. The antibody or portionthereof of claim 95 which is a Fab fragment.
 109. A compositioncomprising the antibody or portion thereof of claim 95 and a carrier.110. The composition of claim 109, wherein the antibody or portionthereof is a monoclonal antibody.
 111. The composition of claim 109,wherein the antibody or portion thereof is a chimeric antibody.
 112. Thecomposition of claim 109, wherein the antibody or portion thereof is ahumanized antibody.
 113. The composition of claim 109, wherein theantibody or portion thereof is a human antibody.
 114. The composition ofclaim 109, wherein the antibody or portion thereof is a single chainantibody.
 115. The composition of claim 109, wherein the antibody orportion thereof is a Fab fragment.
 116. An isolated cell that producesthe antibody of claim
 95. 117. A hybridoma that produces the antibody ofclaim
 95. 118. A hybridoma that produces the antibody of claim
 102. 119.A method of detecting hESF II protein in a biological sample comprising:(a) contacting the biological sample with the antibody or portionthereof of claim 95; and (b) detecting the hESF II protein in thebiological sample by its binding to the antibody or portion thereof.120. The method of claim 119 wherein the antibody is a monoclonalantibody.
 121. The method of claim 119 wherein the antibody is apolyclonal antibody.
 122. The method of claim 119 wherein the antibodyis a chimeric antibody.
 123. The method of claim 119 wherein theantibody is a humanized antibody.
 124. The method of claim 119 whereinthe antibody is a human antibody.
 125. The method of claim 119 whereinthe antibody is a single chain antibody.
 126. The method of claim 119wherein the antibody or portion thereof is a Fab fragment.